1. Field of the Invention
There is a continuing need for accurate and efficient methods for the rapid analysis of small quantities of organic compounds. Such need is related to widely different areas requiring the determination of minute quantities of organic materials. The need to assay diverse substances, from pollutants in water, soil or air which may be present in extremely small quantities to drugs or naturally occurring physiologically active materials, in body fluids, such as blood, urea and saliva, exemplifies the vast array of siutations in which determinations of minute quantities of material are required.
More specifically, both as a medical and police function, the abuse of narcotics and drugs requires an easy method for rapid detection of the use of such drugs, either immediately after ingestion or injection or frequently, after a relatively extended period. The assay should be effective either for the drug, its metabolite, or both, individually or together, and should be specific for the drug being assayed, and not be interfered with by other materials which may be present in the body fluid.
Where the body is malfunctioning, it may be important to assay for particular compounds or metabolites, so as to diagnose the particular malfunction. Also, in case of poisonings, an easy and rapid method for determining the toxin, could be extremely important in providing the antidote.
A wide variety of methods exist for analyzing for a broad spectrum of different organic compounds. Many of these methods are dependent upon different types of detection instruments, such as fluorometers, ultraviolet spectrophotometers, gravimetric analyses, titrimetric analyses, etc. Other methods depend on thin layer chromatography, which is frequently slow, is subject to interference, and may not be reproducible. Because of the significant differences in the procedures, accuracies, and the presence of interfering materials, many diagnostic tests cannot be routinely carried out because of expense and lack of equipment.
2. Description of the Prior Art
The use of free radical probes with naturally occurring materials is found in U.S. Pat. Nos. 3,489,522 and 3,453,288. Labeling of various high molecular weight proteins is described in U.S. Pat. No. 3,481,952. See also Hubbell, et al, Proc. Nat Acad. Sci. U.S., 61, 12 (1968). Organic free radicals have been joined with antibodies and studied. L. Stryer and O. Hayes Griffith, Proc. Nat. Acad. Sci. U.S. 54, 1785 (1965); and J. C. Hsia and L. H. Piette, Arch. Biochem. and Biophys., 132, 466 (1969). In the latter reference, dinitrophenyl antibodies were labeled with 2,4-dinitrophenyl spin labels and the changes in the electron spin resonance (ESR) spectrum were observed as a result of the interaction between the labels and antibodies.
Steroids have been spin labeled by either preparing the oxazole of the 3-keto steroid and oxidizing the nitrogen to the nitroxide or using a carboxyalkyl at the 17 position to form the amide of a tetramethyl(amino)piperidino-oxyl group. See McConnell, et al., Quart. Rev. of Biophys., 3 91-136 (1970); and Hamilton, et al., Structural Chemistry and Molecular History, W. H. Freeman & Co., San Francisco, California (1968), the chapter on spin labels. See also Hubbell, Proc. Nat. Acad. Sci. U.S. 63, 16 (1963).
See also copending application Ser. No. 794,008, filed January 27, 1969, now abandoned, which discloses the use of nitroxide free radical compounds in determining the changes in pH.